1. Field of the Invention
The present invention relates to novel methods for identifying fungal pathogens in a biological sample. In particular, this invention relates to methods for screening biological samples for the presence of fungal pathogens using hybridization methods and probes capable of sensitively and specifically detecting and distinguishing nucleic acid sequences unique to fungi. Also provided are antibodies capable of binding selectively to fungal proteins.
2. Background of the Invention
Candida albicans, once considered a relatively minor fungal pathogen, has recently become a particularly serious health concern as the causative agent of candidosis (also called candidiasis). The incidence of C. albicans infections is rising rapidly with the increase in immune deficiency diseases and immunosuppressive therapy (Bodey and Fainstein, In Systemic Candidiasis, pp. 135 (Eds., Raven Press, New York 1985). Candidosis is a common nosocomial infection afflicting both immunosuppressed and postoperative patients. (Holmes, A. R., et al. Yeast-specific DNA probes and their application for the detection of Candida albicans, J. Med. Microbiol., 37:346-351 (1992)). Although candidosis is a particular concern among immunocompromised individuals, Candida infections are not limited to this group. C. albicans is the major opportunistic fungal pathogen in humans (Odds, F. C., In Candida and candidosis, (Ed.) Leicester University Press, Leicester, United Kingdom (1989)) and is capable of establishing infection whenever the host immune system or normal flora are perturbed.
Although the C. albicans species is a particular health concern, other species of the Candida genus are also pathogenic. The genus Candida is comprised of approximately 200 diverse yeast species classified together due to their lack of a sexual cycle (Meyer et al., In Genus 4, Candida, pp. 1-12, (Ed.) N. J. W. Kregervan Riij, Elsevier, Amsterdam (1984)). A minority of Candida species are pathogenic and 80% of the clinical isolates are either C. albicans or C. tropicalis (Hopfer, R. L. In Mycology of Candida Infections, G. P. Bodey, and V. Fainstein (eds.), Raven Press, New York (1985)).
In immunocompromised hosts, candidosis is a life threatening condition. The prognosis for a patient infected with C. albicans can be improved markedly, however, with prompt antifungal treatment. Treatment may be delayed until a positive diagnosis of Candidosis is obtained since antifungal drugs are toxic. See Holmes, et al., 1992.
Diagnostic tests for the identification of C. albicans or other fungal pathogens in vivo often require complete cultural identification protocols (Musial et al., Fungal Infections of the Immunocompromised Host: Clinical and Laboratory Aspects, Clin. Microbiol. Rev. 1:349-364 (1988)). Methods currently used for the diagnosis of fungal pathogens include: cultural identification, biopsy, serodiagnosis, identification of metabolites, isoenzyme determination, pulsed field gel electrophoresis and analysis of restriction fragment length polymorphisms. Most of these methods are time consuming, laborious and provide inconclusive results. Serodiagnosis is particularly unacceptable for the identification of candidosis, as most individuals have been exposed to Candida and therefore have circulating antibodies against Candida even in the absence of infection. Thus, serodiagnosis can only be accomplished by determining a rise in the titer for anti-Candida antibodies as compared to the titer present in the non-disease state. Such titers are generally unavailable, rendering the technique of serodiagnosis less attractive for the diagnosis of Candida infection.
Potential methods for diagnosing fungal infections through DNA screening have focused on detecting specific nucleotide sequences such as ribosomal DNA (Hopfer, R. L. et al., Detection and differentiation of fungi in clinical specimens using polymerase chain reaction (PCR) amplification and restriction enzyme analysis, J. Med. Vet. Pharm. 31:65-75 (1993)) and the P.sub.450 genes (Buchman, T. G. et al., Detection of surgical pathogens by in vitro DNA amplification. Part I, Rapid identification of Candida albicans by in vitro amplification of a fungal specific gene. Surgery, 108:338-347 (1990)). However, no commercial diagnostic techniques embodying methods related to the identification of these genes in biological samples are known.
One impediment to developing nucleic acid based screening techniques for Candidosis is that basic information about uniquely fungal metabolic pathways and cognate genes of C. albicans is lacking (Kurtz et al., Molecular Genetics of Candida albicans, pp. 21-73, Kirsch, Kelly and Kurtz (eds.) CRC Press Inc. Boca Raton, Fla. (1990)). The sequences of approximately forty C. albicans genes are available in computerized databases, and very few are involved in amino acid biosynthesis. The relatively small database of genetic information available for C. albicans places limitations upon the number of DNA sequences that can be used as targets for screening probes and concomitantly reduces the likelihood of identifying a sequence unique to fungi and amenable to identification through DNA screening techniques. For example, very few of these genes are involved in amino acid biosynthesis.
Similar impediments exist to developing immunological methods of identifying a fungus present in a biological sample. Relatively few antigenic determinants unique to fungi are known, and none are believed to have been successfully utilized as targets for antibody binding in commercially available form.
Among the proteins that have been studied in C. albicans and other pathogenic fungi are the enzymes that make up the .alpha.-aminoadipate pathway for the biosynthesis of lysine. This unique pathway has been identified in Phycomycetes, Euglenids, yeasts and other higher fungi (Bhattacharjee, The .alpha.-aminoadipate Pathway for the Biosynthesis of Lysine in Lower Eukaryotes, CRC Critical Rev. in Microbiol. 12:131-151 (1985); Lejohn, Enzyme Regulation, Lysine Pathways and Cell Wall Structures as Indicators of Evolution in Fungi, Nature 231:164-168 (1971); and Vogel, Two Modes of Lysine Synthesis Among Lower Fungi: Evolutionary Significance, Biochim. Biophys. Acta 41:172-174 (1960)) and is present in C. albicans and other pathogenic fungi (Garrad, R. Masters Thesis, Miami University (1989) and, Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans, J. Bacteriol. 174:7379-7384 (1992)). Lysine is an essential amino acid for humans and animals and is synthesized by the diaminopimelic acid pathway in bacteria and plants. The .alpha.-aminoadipate pathway consists of eight enzyme catalyzed steps; there appear to be seven free intermediates in S. cerevisiae (Bhattacharjee, The .alpha.-aminoadipate pathway for the biosynthesis of lysine in lower eukaryotes, CRC Critical Review in Microbiol. 12:131-151 (1985)). The final reversible step of the .alpha.-aminoadipate pathway is catalyzed by saccharopine dehydrogenase (EC 1.5.1.7), which is encoded by the LYS1 gene of S. cerevisiae and C. albicans , and the LYS5 gene of Y. lipolytica (Fujioka, Chemical mechanism of saccharopine dehydrogenase (NAD, L-lysine forming) as deduced from initial rate pH studies, Arch. Biochem. Biophys. 230:553-559 (1984); Garrad and Bhattacharjee, Lysine biosynthesis in selected pathogenic fungi: Characterization of lysine auxotrophs and the cloned LYS1 gene of Candida albicans, J. Bacteriol. 174:7379-7384 (1992); and Xuan et al., Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica, Mol. Cell. Biol. 10:4795-4806 (1990)).